Controlled Release of Recombinant Proteins
With the rapid advances made in biotechnology and genetic engineering, a growing number of proteins and peptides have been produced by recombinant DNA technology for use as pharmaceutical therapeutic agents. Such proteins include erythropoietin (EPO), granulocyte-colony-stimulating factor (G-CSF and GM-CSF), interferons (alpha, beta, gamma, and consensus), insulin and interleukin-1 etc. In addition to these proteins, several hundred other proteins are currently undergoing clinical trials as drugs. Because proteins have generally short in vivo half-lives and negligible oral bioavailability, they are typically administered by frequent injection, a procedure that is hard to accept for most patients. It has been conjectured that the employment of controlled release technology for the administration of such therapeutic agents can alleviate such a problem to a degree. Hence, it is highly desirable to develop sustained-release systems.
Tissue engineering is another area of biomedical research that is much pursued. The goal of tissue engineering is to employ the techniques of modern biotechnology to regenerate or replace lost or damaged tissues and organs. In addition to cells and their scaffolds, a class of proteins called growth factors are frequently required for tissue engineering. These include nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), tumor necrosis factor (TNF) etc. Growth factors, most of which are globular, are effective in the process for supplying the oxygen and nutrients which are necessary for the survival of the transplanted cells in organ transplants (Lanza et al., 2000).
Cell growth factors are usually injected locally and active targeting of the agent is not absolutely necessary. However, it has been found that direct single-time injection of a growth factor solution into a regeneration site is less effective, as the injected growth factor rapidly diffuses away from the site. Repeated injection is, of course, inconvenient. Novel drug delivery systems are thus desired. The projection is that controlled delivery systems for recombinant proteins will be a major technology in tissue engineering during the next century. (Yasuhiko, (2000) Pharmaceutical Science & Technology Today, 3: 80–89)
Microparticles as Controlled-release Carriers
Microparticles has held promises in many areas of medicine. In recent years, microparticles have garnered growing attention and have been the subject of investigation as an ideal drug carrier. Up to the present, microparticles have found application in more than thirty different drugs, including antipyretic analgesic, antibiotic, fibrin and anticancer drugs, etc. Biomolecules such as proteins, enzymes, hormones and peptides, are sensitive and easily degraded. The most promising controlled-release approach would be to encapsulate such materials within microparticles. A principal advantage of formulating these sensitive biomolecules in microparticles is that they may be administrated by injection, and does not require formal surgical procedure for their administration.
Materials that are useful for making into microparticles can be grouped into three categories: natural polymers such as glutin, alginate and chitosan; semisynthetic polymers such as carboxymethyl cellulose, cellulose acetate phthalate, methyl cellulose and ethyl cellulose; and synthetic polymers such as polyamide, poly(acrylic acid), poly(vinyl alcohol), polycarbonate, poly(amino acid), poly(lactic acid), poly(lactide-co-glycolide) and poly(d,l-lactide)-poly(ethylene glycol) copolymer. Natural polymers are abundant and usually biodegradable. However, the principal disadvantage is in the difficulty of their modification and purification. There are significant batch to batch and source to source variations, due to the need to isolate these materials from living organisms.
The requirements for materials used for encapsulation are suitable drug release rates, stability, non-toxicity, absence of interference with the pharmacological action, strength, suitable hydrophilicity, plasticity, permeability and solubility. In addition, it would be desirable to have microparticles made from biodegradable polymers to eliminate the need for their removal after the agent has been released. Synthetic polymers are available in a wide range of compositions with readily adjustable properties. Therefore, much attention has been paid to the use of biodegradable materials. Synthetic polyesters have, especially, been widely investigated. See, Blanco et al., (1998) Eur. J. Pharm. Biopharm. 45: 285–294, Zhu et al., (1999) Eur. Polym. J. 35: 1821–1828.
Up to the present, most researchers have concentrated on the use of hydrophobic biodegradable polymers. Singh et al., (2001) J Control. Release 70: 21–28, reported the in vitro and in vivo release behavior of polylactide-co-glycolide microparticles with entrapped insulin growth factor (rhIGF-I). Jain et al., (2000) Eur. J. Pharm. Biopharm, 50: 257–262, investigated the release behavior of bovine heart cytochrome C and heart skeletal muscle myoglobin from injectable PLGA microparticles.
There are various problems associated with the applications of such polymers. The problems are: the difficulty of the homogeneous dispersal of the hydrophilic drug within the polymer matrices, the inability of certain macromolecules to diffuse out through the polymer matrix, the unpredictability of drug release behavior, the deterioration of the drug, e.g., denaturation caused by the presence of organic solvents, and irritation to the organism due to side effects caused by the presence of organic solvents.
Degradable polymers containing water-soluble polymers have also been investigated. Copolymerization of lactide, glycolide and caprolactone with the polyether such as polyethylene glycol (PEG) was expected to partially overcome the above drawbacks, while taking advantage of the virtues of both biodegradable and hydrophilic polymers. Sawhney et al., (1990) J Biomed. Mater. Res, 24(10): 1397–1411, Casey et al., (U.S. Pat. No. 4,716,203) describes the synthesis of a block copolymer of PGA (poly(glycolic acid)) and PEG. Cho et al (2001) J. Control. Release 76: 275–284, applied the W/O/W double emulsion method to prepare PLLA-PEG copolymer microparticles, where bovine serum albumin (BSA) was used as the model drug. Although the copolymers have improved hydrophilicity, most of the biodegradable synthetic polymers reported so far can only be processed in organic solvents which are harmful to protein activity. For these reasons, it is desirable to have a hydrogel as a preferable candidate as a protein drug carrier.
Use of Hydrogel as Protein Carriers
Hydrogels have been intensely investigated as protein drug vehicles because of their excellent biocompatibility and hydrophilicity. Compared with other synthetic biomaterials such as PLGA, hydrogels more closely resemble natural living tissues because of their high water contents and soft and rubbery consistency. The nature of hydrogels minimizes irritation to surrounding tissues. Furthermore, hydrogels are useful in protecting the drug from hostile environments, e.g., the presence of enzymes or the low pH in the stomach. Some biodegradable hydrogels have been reported, Sawhney et al., (1993) Macromolecules, 26:581–587, and Hubbell et al., U.S. Pat. Nos. 5,986,043, 6,060,582, and 6,306,922. However, the hydrogels were synthesized via ultraviolet polymerization or photopolymerization, which are not suitable for entrapping ultraviolet-sensitive proteins.
There are many different physical forms of hydrogels, such as microgel, bulk gel etc. Since microparticles are injectable, microgel would be a good carrier for proteins. Drug delivery systems in the form of microparticles may enable the release of the therapeutic agent in a specified area or over a specified time period. Kim S W et al., (1997) Nature 388: 860–862, studied injectable hydrogel. During the process of loading drug, the use of any organic solvent which can denature the protein was avoided. However, in this case, bulk gels instead of gel particles were used, and the encapsulation temperature was higher than human body temperature.
Cross-linked glutin and collagen have also been employed as a hydrogel to encapsulate peptides of opposite charges. Alginates have been shown to be able to encapsulate biological materials. Lim, U.S. Pat. No. 4,352,883. But the rates of degradation of both kinds of gels were not easily controlled over a wide range of conditions.
In addition to swelling, some hydrogels also show changes in response to stimuli. The present inventors explored these characteristics for developing novel ways for drug loading and drug release.
There have been many studies on materials for drug release. These materials are either merely biodegradable as reported by Cohen et al., (1991) Pharm. Res., 8: 713–720, Langer et al., (1998) Nature 392: 5–10, Bawa et al., (1985) J. Control. Release, 1: 259–267, Li et al., (2002) J. Polym. Sci. part A: Polym. Chem. 40(24): 4550–4555, Fu J. et al., (1997, 1998) Chemical Journal of Chinese Universities, 18(10):1706–1710,19(5):813–816; or merely responsive to environmental stimuli, see e.g, Wu et al., (1995, 1996) Macromolecules 28(15): 5388–5390, 29(5):1574–1578, Wu, U.S. Pat. No. 6,030,634; Zhou et al., (2003) J. Polym. Sci. part A: Polym. Chem. 41(1):152–159. There have been a few reports about a material having a combination of both properties, Shah, U.S. Pat. No. 6,541,033, and Shah et al., U.S. Pat. No. 0,099,709. From these few reports, the use of biodegradable and thermosensitive microgels in a drug delivery system is still quite limited.
RealGel™ is a block copolymer with temperature-sensitivity and degradability, Zentner et al., (2001) J. Control. Release, 72: 203–215. It is not a chemical gel but a physical gel. The term of “chemical gel” represents a gel in which gellation is due to chemical crosslinking, while the term of “physical gel” denotes a gel in which gellation is induced by physical parameter such as temperature. In addition, this gel is a liquid at high temperature, and forms a semi-solid gel when it can be used to encapsulate a drug at a lower temperature. However, the positive temperature sensitivity may lead to denaturation of the protein when it is being encapsulated at a high temperature before gelation. A negative temperature sensitive biodegradable chemical hydrogel has been reported by Hubbell et al., (2000) U.S. Pat. No. 6,129,761, but it was a bulk gel, wherein an ultraviolet initiation method was used. There are difficulties in applying this type of bulk gel in an inverse suspension polymerization process.
In accordance with the present invention, a thermosensitive, biodegradable microgel for the sustained delivery of drugs is provided. The drug is released at a controlled rate from the microgel, which eventually biodegrade into non-toxic products. The rate of degradation can also be adjusted by adjusting the composition of the biodegradable groups.
Drug Loading
In order to encapsulate protein molecules within microparticles, most researchers utilize a solvent evaporation method because this method is useful for achieving a high level of encapsulation. There are basically two different approaches for encapsulation: a water/oil/water double emulsion or a single emulsion in which the micronized protein powder is dispersed into an organic solvent phase containing the dissolved polymer. However, many proteins are irreversibly denatured by contact with organic solvents necessary for dissolving the polymer. Thus, one of the main problems of such methods is the partial or complete loss of biological activity. There are some additives, such as albumin and polysaccharides, which can be used to stabilize the proteins to a certain degree and affect the release behavior, Baldwin et al., (1998) Adv. Drug. Deliv. Rev., 33: 71–86. The alternative, but less frequently reported method is loading the proteins or peptides into a microgel by swelling the microgels in an aqueous solution and subsequent drying. However, this method provides only a low level of encapsulation efficiency. Thus, a further object of the present invention is to provide a process of manufacturing a microgel and loading a protein drug into the microgel with a relatively high loading level.
Compared to in situ encapsulation of the drug, absorption of the drug AFTER the preparation of the gel or hydrogel provides several striking advantages: (1) The biocompatibility of the polymeric material may be enhanced because gel without protein loaded can be easily cleaned to wash out any residual monomers, initiators etc.; (2) the steps of preparing the microgel followed by encapsulation of the protein are separate steps, making it much easier to control each of the two steps independently. On its face, it has been considered impractical to adsorb the proteins after the preparation of the gel or microgel. The reasoning was that if a drug can penetrate the gel easily, it would not be released slowly; whereas, if drug release can be controlled well, it would be hard to adsorb the drug into the gel after gel formation and would lead to a very low loading level.
In accordance with the present invention, the hydrogels of the present invention have been designed to be “intelligent” to overcome the difficulties encountered previously. The “intelligent” hydrogels of our invention comes from the temperature sensitivity of the polymeric material in a particular solvent media. The gel swells at a low temperature and is ready for drug absorption at this low temperature. However, the gel contracts or gelates at a higher temperature, such as the body temperature, and provides a unique way for controlling the release of the drug after injection into the body.
The object and features of the present invention will be made apparent from the following description.